Deletion analysis of the NS1-NS2A region of the dengue virus polyprotein indicated that a minimum length of eight amino acids at the NS1 C-terminus is required for cleavage between non-structural proteins NS1 and NS2A to take place. Comparison of this eight amino acid sequence of dengue type 4 virus with the analogous sequences of other flaviviruses identified a sequence motif in which Ala(-1), Val(-3), Ser(-5), Val(-7), and Met/Leu(-8) are conserved. Amino acids at other positions vary. We were interested in determining whether the observed sequence motif is optimal for cleavage of the polyprotein at the NS1-NS2A junction. Mutants of NS1-NS2 were constructed that contained an amino acid substitution at different positions upstream of the cleavage site or at the immediate downstream position. The effect of these amino acid substitutions on cleavage of NS1-NS2A was then analyzed. The results showed that most substitutions of Ala(-1) or Val(-3) yielded predominantly uncleaved NS1-NS2A fusion protein. On the other hand, replacement of Thr(-2) had little or no effect on cleavage. These results are in agreement with the proposed consensus sequence deduced by comparison of sequences among flaviviruses. Several substitutions of Gly(+1) yielded reduced cleavage indicating that Gly at this position also plays a role in cleavage. Mutants that contain a single amino acid substitution in one of the remaining five positions have been constructed, and work is in progress to define their NS1-NS2 cleavage phenotype. A panel of amino acid substitution mutations that had a varying effect upon efficiency of NS1-NS2A cleavage were selected for incorporation into the full-length cDNA, and in vitro derived RNA transcripts were used to transfect cells and recover viable dengue virus mutants. These viable mutants exhibited growth restriction as revealed by plaque analysis. Evaluation of other viral growth properties of these mutants is in progess.